Recent technological advances in cryo-electron microscopy (cryo-EM) have led to unprecedented resolution for native-state (unfixed, unstained) macromolecules and cells. Amino-acid side-chains can now be identified in maps of isolated macromolecules, without the need for crystallization. The in-situ shapes of important macromolecules within cells, and their cellular interaction, can now be studied at down to 8.5 resolution by cryo-EM tomography. These advances promise insight into details of cellular and molecular function that have never before been accessible, advancing all fields of biomedical research; NIH has recognized their importance by a new RFA (GM-16-001) that aims to widen access to this technology. However, current enthusiasm will wane with the realization that the full promise of this technology is currently only achieved for certain macromolecules or cell types. The PI's lab focuses on two projects that will widen applicability of advanced cryo-EM: (1) cryo-EM phase-plate imaging and (2) cryo-focused-ion-beam (cryo-FIB) preparation for cryo-EM tomography. The highest resolution in cryo-EM of isolated macromolecules requires molecules of sufficient size (> ca. 100 kDa), limited flexibility, ideally with some symmetry, and which form a fairly homogenous population. Without increasing electron-beam damage, phase-plate imaging greatly improves contrast, so that adequate selection, sorting, and alignment of particles can be accomplished, using fewer total particles. This will take us along the last mile to a wider application of the improved single-particle approach. We will continue to evaluate a wide variety of phase-shifting devices, and improve their use in a variety of real-world situations. The highest resolution for in-situ macromolecules is achieved by cryo-EM tomography followed by sub-tomogram averaging of molecular motifs. Samples must be thin; flat-cultured cells were previously examined only at their margins. For cells in tissue, cryo-ultramicrotomy, a difficult and artifact-prone technique, was the only choice. We introduced cryo-FIB milling, which can facilitate high-resolution results with all cell types. But in spite of recent progress, this remains an extremely difficult technique, so far implemented in only very few labs in the world. In addition, targeting specific sub-cellular structures is problematical. Ifthe promise of cryo-FIB milling for cryo-EM tomography is to be realized, it must be refined to widen its applicability and make it easier to use. Cryo-EM tomography will also greatly benefit from cryo-EM phase-plate imaging; better contrast will improve tilt-series and sub-tomogram alignment accuracy, and will reveal weak fine structure. Our lab has built up world-class instrumentation and infrastructure specific to our projects (likely unmatched elsewhere), and we have personnel with many years of experience in advancing cutting-edge biological electron microscopy. With funding via the MIRA concept, we aim to bring our current projects to full fruition, while remaining in a position to follow exciting new developments.